Ligand receptor-ligand assays on solid surfaces, such as sandwich assays, have been a standard tool in chemical and biological research and in diagnostics for many decades. For instance, Enzyme-Linked Immunoabsorbent Assays (ELISA) have been standard for quantitative analysis of protein in blood and other body fluids. Assay results have often been read by quantitative measurement of fluorescence from tags associated with ligand receptor-ligand complexes on the solid surface.
In the recent decade there has been a strong effort to perform multiple ligand receptor-ligand assays simultaneously within a small geometry, in order to reduce reagents, labor and sample sizes. In the context of genetic research and elsewhere, especially where qualitative results are all that are required, microarray technology employing spotted arrays within portable cassettes has been adopted for the assays.
However, significant obstacles have confronted development of miniaturized quantitative assay formats that are both highly sensitive and highly reproducible, for which there is great need. The obstacles have been even greater when further desired features are sought, such as simplicity, rapidity, economy and broad range applicability.
In particular, in the area of protein analytes, significant obstacles have confronted development of miniaturized, multiplexed immunoassays that enable direct comparison to or substitution for the results of standard ELISA of analytes in body fluids, especially, blood. For qualification of drugs for human use and for medical diagnostics, such assays are required to be very precise, preferably with a coefficient of variation (CV) less than 10. (The coefficient of variation (CV), defined as the percent ratio between the standard deviation of a set of values from repeated testing of a given sample and the mean of those values, provides an estimate of the degree of variability among those values. The lower the CV, the more precise the assay and the more confidence one has in the results.)
Without attempting to be exhaustive in listing the obstacles to the development of precise miniaturized assays, a few examples will be mentioned.
Some workers in the field have suggested that conditions of equilibrium are prerequisite for achieving high sensitivity and low coefficient of variation in an assay. But, use of very small samples is particularly difficult in an equilibrium assay, because such an assay is generally volume-dependent. Accuracy and repeatability depend on controlling the accuracy of the sample volume. Unfortunately, when the sample volume is very small, an assay that depends on maintaining an accurate sample volume is prone to variation from assay to assay. Even if the sample size is controlled by an automated metering system, there is such variation.
Another proposal has been to detect analytes by a method based on an ambient analyte theory that requires particularly small spots of ligand receptors. This size requirement challenges the capabilities of conventional instruments used in producing spotted microarrays. Furthermore, very small spots present the difficulty, when reading the results, of lack of sufficient resolution and of sufficiently low background noise. Furthermore, such assays take long to perform, typically well over one hour.
Various approaches to the design of miniaturized multiplexed assays have employed microfluidic technology, with flow channels and reaction chambers having flow cross-section typically less than 0.02 mm2. Numerous uncontrolled phenomena can influence the results in such a system. Liquid temperature change, as well as atmospheric pressure change, and localized liquid pressure change as liquids encounter sharp edges, space discontinuities, or variation of surface properties such as surface roughness or different surface tension properties, can all produce gas micro-bubbles that, we have realized, have heretofore not been adequately recognized or taken into account in the context of assay cassettes.
Other approaches, such as those employing capillary or osmotic forces for moving liquids through an assay, have been highly sensitive to the characteristics of the particular fluids and passage materials involved, and have required custom development for narrow groups of analytes. With some such approaches, a requirement of intersections with discrete lanes of migrating liquid has limited the amount of data obtainable in a desirable geometry and has prevented achieving desired coefficient of variation in quantitative results.
Still other approaches have suffered from complexity, high cost, requirement of skilled operators, or lack of suitability for implementation in disposable cassettes.
In general, the ability to measure analyte concentrations in miniaturized, multiplex assays suffers from what has appeared to be necessary tradeoffs between sensitivity, repeatability, performance, cost and ability to compare results with standardized assays.
Developers of biological assays have made various attempts to escape some of the constraints associated with conventional assay techniques. An example is by reading an assay by electrical measurement. Effort has been expended in the development of chemical sensors that can measure the presence or the concentration of chemical species in blood or other biological fluids in this manner. One of the drawbacks of such methods is the inability to enable direct comparison to standard ELISA results. Other drawbacks are the requirement of accurately controlled liquid volume, and the difficulty of performing the assay on multiple analytes simultaneously. Besides, there is the drawback of having to make electrical connection with an assay cassette; over time, the electrical terminals may introduce inaccuracies or require maintenance. Designs of this category, as well, may suffer from uncontrolled phenomena mentioned above, to which aspects of present invention offer solution.
In view of the limitations and drawbacks of prior approaches, there is considerable need for improved approaches to the problems of miniaturized quantitative assays using arrays on solid surfaces, both with respect to assays that employ ligand receptor-ligand systems, very generally, and in particular, with respect to immunoassays, especially immunoassays for protein in body fluids such as blood.
There is special need for miniaturized assays that can be analyzed using fluorescent detection and are otherwise comparable to standard ELISA results. There is particular need for sensitive, precise multiplex assays to enable simultaneous evaluation of multiple markers that may indicate disease, the effectiveness of a drug, toxic reactions to a drug, or suitability of subjects as candidates for a drug or drug study.
There is particular need for an assay technique (or “platform”) for protein biomarkers which, above all, is robust. An assay technique is needed that produces highly credible results with low coefficients of variation, is immune to interferences, and performs its function with sufficient margins that it is useful over a wide range of analytes, levels of dilution, and starting conditions. It is especially desirable that the technique also be sensitive, have significant throughput, be capable of conducting multiple assays at once and employ equipment which is easy to use. It would be beneficial, as well, for such a technique to enable simple design of particular assays and to require little sample or reagent and little handling of the sample.